Duke University | Howard Hughes Undergraduate Program

Don’t you just hate it when 3 days worth of work goes down the tube? Better yet, don’t you hate it when you follow a protocol (which you have successfully done twice before) to the letter, and you get still get nothing?

That’s basically what I endured yesterday. This Tuesday, I was running smoothly through my protocol for nuclear translocation assays (again testing for the presence of “Protein X”). I did everything that I had done before with this assay…starving the endothelial cells, restimulating them with a particular ligand, permeablizing their membranes so that antibodies can enter, stimulating them with the specific antibody for “Protein X”, and finally adding a secondary antibody with a fluorescent green tag. All stuff I’d done before.

Except THIS time, when I looked under the microscope…nothing. Absolutely nothing. No green glow from the fluorescent tag. Same problem for all 12 slides I look at. I won’t bother to put up a picture, because that’s what you would see…nothing. Blackness.

So yeah, it was a pretty frustrating experience, but that’s the best way to learn. After some troubleshooting, me and Nam concluded that the secondary antibody (the one with the fluorescent tag) was the problem. Or so we think. Tomorrow (Friday) after letting my cells grow, I’ll stick a different antibody on “protein X”, this one with the old red tag that I had used earlier.

It could have been the antibody. It could have been contamination. Or, it could have been me being careless. But regardless, like I said, messing up is definitely the best way to learn, even though it is pretty frustrating. The best way to get better at something is to practice, and this is no exception.

Til next time.

“The greatest mistake you can make in life is to be continually fearing you will make one.”
-Elbert Hubbard
 

Questions and Answers

At this point, I’m starting to get a better idea of my “research question” for the summer, or in other words, what I am trying to answer through my research and experiments here in the Blobe Lab. I think it could go something like:

“In endothelial cells, how do various stimuli, in the form of ligands, affect the nuclear translocation of “Protein X” in the presence and absence of the TGF-Beta pathway co-receptor, endoglin?”

If you don’t understand my question, check out some of my earlier posts. I think it’s still too early for me to have any definite hypotheses, partly because Nam and I are going into territory that really hasn’t been explored before. There are also a number of experimental parameters we will need to play around with before we get major results. I’ll have to spend a LOT more time running my translocation assays and looking under the fluorescent microscope before I start to see any major trends. But that’s science I guess. Patience is key.

Here's a picture of some endothelial cells from another nuclear translocation assay I ran the other day. Again, I'm staining for the translocation of "protein X". This time, I used a fluorescent green antibody tag:

“Patience is something you admire in the driver behind you, but not in one ahead.”
-Bill McGlashen
 

So, it turns out that the project I told you about last time, the one about SMAD translocation and such, won’t be what I will pursue this summer. After conducting nuclear translocation assays testing for the presence of SMADs in the nucleus, we didn’t get the results we would have liked. So, we’re going to put that project on the back burner for now.

Instead, I’ll be working on a similar project involving the nuclear translocation of a different protein…let’s call it “protein X”. This project is similar to the old one because it involves the same experimental techniques and protocols. Basically, we are trying to see whether “protein X” translocates into the nucleus in the presence of certain other molecules. “Protein X” has recently been found to be extremely important in cancer research because of its potential to regulate the cell cycle. Specifically, “protein X” is seen to have anti-apoptotic tendencies. In other words, when activated, “protein X” can prevent cell-mediated death and improve a cell’s reproductive abilities. “Protein X” does this by negating the effects of proteins that promote cell death, and conversely, enhancing the effects of proteins that promote cell proliferation. Nuclear translocation of “protein X”, like the SMADs, is important because “protein X” can regulate cell development through interaction with transcription factors in the nucleus. Again, these transcription factors genetically affect a cell’s development, similar to what happens in the SMAD pathway.

If you’re confused by what I’ve said so far, it’s alright. By no means, at this point, do I understand 100% of exactly what we’re doing. But the most important thing we can do, as Nam has told me, is ask questions. You never know when something will click. On that note, I believe that knowing WHY to do something is more important than knowing HOW to do something.

I’ll leave you with a picture of some endothelial cells I looked at under the fluorescent microscope today. Each one of those red dots you see is a fluorescent antibody “tag” that represents the location of “protein X.”

“Success depends upon previous preparation, and without such preparation there is sure to be failure.”
-Confucius
 

What do I expect from being in a research lab for 8 weeks this summer?

This summer I hope to have a very fulfilling research experience that will help me down the road. The more experience you have in a lab, the better, in particular if you are looking into a research career in the future. The more time you spend in a lab, the more you will learn about the protocols and techniques that are applicable to any lab environment. For example, last year in the Benfey lab, I learned about the basics of “sterile technique”, or in other words, how to prevent contamination from occurring when conducting an experiment. Good sterile technique can only come through practice, and obviously you get practice by spending more time in the lab.

From a more practical standpoint, the more time you spend in a lab, the more you are exposed to universal experimental procedures. For example, last year I became pretty proficient at conducting gel electrophoresis and PCR in Dr. Benfey’s lab to test for the presence of genetic material. This summer, I was introduced to the Western Blot, which is used to test for the presence of certain proteins. Although I had never conducted a Blot before, the procedure itself seemed familiar to me because of my previous research experience. Both techniques involved setting up gels and letting them run in an electric current so that the molecules of interest (DNA or proteins) would be separated by their weight. “Running gels” for DNA or protein are similar procedures.

Finally, this summer I hope to work on my own project, for the first time, and hopefully make some useful discoveries for Dr. Lee and the Blobe lab. I realize that because I am working on my own without the years of experience of my mentors, I am bound to make mistakes. But, that’s the best way to learn, after all. Of course, my mentors will still be there to guide my progress and answer my questions.

So you might be wondering about what it is that I will actually be working on this summer, or what my project actually is. I can’t give too much away right now, without knowing the results of an experiment that I ran on Saturday (yep, you have to come into the lab on weekends, if need be). But, here’s a taste.

Basically, in the TGF-beta cell-signaling pathway, which I talked about in an earlier post, there are certain molecules called SMADs that can travel to the nucleus of a cell and act as transcription factors (molecules that affect how genetic information is expressed by a cell). Now, there are many different kinds of SMADs, and they can have different effects when they serve as transcription factors. In cell development, certain transcription factor SMADs can have opposite effects: they can either induce cell death (apoptosis), or they can cause cellular proliferation, and potentially, cancer. So, it’s important to know how these SMADs work, because they can play an essential role in uncontrolled cell proliferation, or in other words, cancer. Other molecules in the TGF-B pathway affect which SMADs are allowed to travel to the nucleus (nuclear translocation) and function as transcription factors. So, we want to see which and how these SMADs move to the nucleus in the presence of certain “other” molecules.

Sorry, no details yet. But soon! :]

And also. This summer, I expect to learn how to cook. The hard way. Recently, me and my roommate, Nick, went on a rampage at the local Target, shopping for ingredients. Here's me, modeling our newly-stocked fridge. Yum.

“Do not go where the path may lead, go instead where there is no path and leave a trail.”
-Ralph Waldo Emerson
 

Points for those of you who got my movie reference. Anyways, I recently asked Dr. Blobe, the head of the Blobe Lab, and Nam Lee, my research mentor, a few questions about who they are and what they do. Take a read:

What got you interested in research in the first place?
Dr. Blobe: He has always had an interest in science. Having good mentors always helps. He started research in middle school, and really got interested after his freshman year of college.
Nam: “High school. Science was what I excelled in.”

Could you tell me a little something about your undergraduate and graduate
school education? Where did you go, and what did you do? Were you involved
in research as an undergraduate, or even as a high school student?
Dr. Blobe: As an undergrad, he attended Notre Dame, majoring in chemistry. He was in the marching band, and did a lot of research in his spare time.
Nam: “I was involved in a Howard Hughes summer biology program in high school. I
went to U of Iowa for both undergrad and grad school. As an undergrad I
started working part time in a biochemistry lab.”

Why are you interested in better understanding cell signaling and the
TGF-beta pathway in particular?
Dr. Blobe: Most human diseases could be linked to signaling gone bad, and we need more information about how the TGF-beta pathway works.
Nam: “I was always interested in cell signaling, and TGFbeta pathway suited my
research interests as a postdoc.”

In your opinion, what is the most gratifying aspect of the research
process, and why?
Dr. Blobe: He has 2 favorite aspects: 1) Getting to see the growth of scientists. He likes to see how students, especially grad students, progress from student to scientist. 2) Participating in novel research and exciting discoveries that could benefit everyone later down the road.
Nam: “Publishing. Research is most gratifying when others get to read and
critique your research. No sense in keeping research data to yourself.”

Is there something you really could do without, in terms of the research
process?
Dr. Blobe: The growing bureaucracy, and all of the red tape. He doesn’t really like all the nit-picky safety protocols, but says it’s a necessary evil.
Nam: “All the meetings.”

What’s something you really like about the people in your lab?
Dr. Blobe: He likes that he has dedicated, good people. He wants them to work hard, play hard, and truly enjoy their work.
Nam: “Different personalities.”

What do you enjoy doing in your free time? Do you have any favorite books,
or music that you really enjoy listening to?
Dr. Blobe: He enjoys spending time with the family. He has 4 kids, 2 grandkids, and enjoys going on vacations whenever he can. He also enjoys a healthy game of poker. His favorite authors are Lee Child, and Michael Connelly.
Nam: “I enjoy drinking wine and cocktails. I read as much as I can, mostly
history books.”

If you could be one kitchen appliance, what would it be, and why?
Dr. Blobe: He’d be a panini maker. It’s fast, versatile, and fun.
Nam: “I would be a refrigerator. I'm that cool.”

They’re both pretty cool dudes. That’s it for now!

“A hero is no braver than an ordinary man, but he is braver five minutes longer.”
-Ralph Waldo Emerson